The use of fusion proteins as a tool for recombinant protein production is well known in the biopharmaceutical industry. Fusing the coding sequence for a desired recombinant protein to that of a well-expressed gene has several advantages. Most fusion protein strategies position the protein of interest at the C-terminal end of the highly expressed fusion partner which allows translation initiation to occur on a "proven" gene sequence that is known to be well translated and can help ensure high expression levels. Some fusion partners can coffer many advantageous attributes to the fusion protein, such as specific cellular localization, binding to affinity ligands to aid in purification and detection, and even proteolytic and conformational stability.
While fusion proteins offer numerous advantages, this beneficial physical association of the protein domains can also be problematic when it becomes necessary to separate the two (or more) components from their covalent tethering. The method of protein cleavage must be both specific and efficient and must not yield unwanted side products. This is particularly so when utilizing a fusion protein approach for the production of biopharmaceuticals destined for human use. Ideally, the most useful method allows for cleavage at a specific target sequence without regard for the internal protein sequence and/or without regard for the composition of the fusion partners. The method should produce cleaved product with authentic N- and C-termini, should not modify or otherwise adulterate the desired protein product, and should be tolerant to a wide range of conditions so that reaction components can be tailored to the physical characteristics of the fusion protein without seriously affecting the efficiency of the cleavage reaction. In addition, for biopharmaceutical production and applications, the cleaving reagent should not be from an animal source due to concerns about contamination by infections agents.
An ideal choice for such a "universal" fusion protein cleaving method is use of the mammalian enzyme enterokinase (enteropeptidase). Enterokinase is the physiological activator of trypsinogen and cleaves with high specificity after the sequence (Asp.sub.4)--Lys (SEQ ID NO: 24). Light et al., J. Protein Chem. 10:475-480 (1991). It is possible to engineer the fusion protein to include a linker DNA sequence encoding the amino acid sequence recognized by enterokinase. See for example, Bollen et al., U.S. Pat. No. 4,828,988 (May 9, 1988); Rutlet, U.S. Pat. No. 4,769,326 (Sep. 6, 1988); and Mayne et al., U.S. Pat. No. 4,745,059 (May 17, 1988). However, although extensive research efforts have been mounted by several different research groups since the first partial purification of bovine enterokinase more than 15 years ago, no one has yet been successful in cloning enterokinase. Porcine enterokinase was first isolated in the early 1970s (Maroux et al., J. Biol. Chem. 246:5031(1971))and bovine (Anderson et al., Biochemistry 16:3354(1977)) and human (Grant et al., Biochem. J. 155:243(1976)) enterokinases were isolated in the late 1970s. Liepnieks et at., J. Biol. Chem. 254: 1677(1979) described an enterokinase having 35% carbohydrate, a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Subsequent studies oft he light chain, i.e., the catalytic subunit, were reported in Light et al., J. Biol. Chem. 259:13195(1984). Most recently, Light et el., J. Protein Chem. 10:475(1991), disclosed what was later proven to be an incorrect partial amino-terminal sequence for the catalytic subunit of bovine enterokinase. To date, it has been impossible to obtain recombinantly produced enterokinase activity and there continues to exist a need for such a product.